Affinity Purification of Multifunctional Polymer Nanoparticles

Abstract

We report that multi functional polymer nanoparticles approximately the size of a large protein can be "purified", on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The selection process takes advantage of the thermo-responsiveness of the nanoparticles allowing "catch and release" of the target peptide by adjusting the temperature. Purified particles show much stronger affinity (Kd app ≈ nM) and a narrower affinity distribution than the average of particles before purification (Kd app > μM) in room temperature, but can release the peptide just by changing temperature. We anticipate this affinity selection will be general and become an integral step for the preparation of "plastic antibodies" with near homogeneous and tailored affinity for target biomacromolecules.General procedures for the creation of synthetic materials with biomacromolecular recognition sites are of significant interest as a route to stable, robust and mass-produced substitutes for antibodies. [1][2][3][4][5][6][7][8] Ideally, recognition of complex biological targets, including proteins, peptides and carbohydrates, requires multiple functional groups that contact target molecules by a combination of electrostatic, hydrogen-bonding, van der Waals, and/or hydrophobic interactions. It has been shown that copolymerization of optimized combinations and ratios of functional monomers creates synthetic polymer materials with molecular recognition sites. [1][2][3][4][5] However, in contrast to antibodies whose exact sequence can be determined and cloned, polymerized materials result in heterogeneous structures with a distribution of recognition sites. 1,3,7 This is an intrinsic property of polymers synthesized under kinetic control, in contrast to the synthetic small molecular hosts prepared by multi step reactions 9 or by self-assembly under equilibrating conditions 10 . Here we demonstrate a general procedure to purify synthetic polymer nanoparticles (NPs) with high-affinity binding sites for a target biomacromolecule from a random pool of multi-functional copolymer nanoparticles (MFNPs). These nanoparticles are approximately the size of a large protein and are "purified" on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The concept of affinity purification of NPs was demonstrated with melittin, a 26 amino acid peptide ( fig. 1a), as the target molecule. Melittin has six positive charges of which four are localized in a hydrophilic six amino acid sequence on the C-terminus. The remaining twenty amino acids have a high proportion of apolar residues. 11For the MFNPs, we chose cross-linked N-isopropylacrylamide NPs (~30 nm) incorporating hydrophobic (N-t-butylacrylamide (TBAm)) and negatively charged (acrylic acid (AAc)) functional monomers ( fig. 1b). We have reported that NPs with this composition interacts with melittin (K dapp = 46 μM) via both electrostatic-and hydrophobi nteractions in PBS (35mM phosphate buffer/0.15 M NaCl, pH 7.3). 5 However, th...

The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo

“…The capture of such toxins would likely require positively charged hydrophobic NPs, a combination that would challenge the immune system. Incorporation of "custom" monomers, designed to bind to specific amino acid or peptide sequences by hydrogen bonding and/or van der Waals interactions, (9), and by applying molecular imprinting (19,29) and/or affinity purification (20) might offer a more fruitful direction to create a synthetic polymer antidote for such toxins. Surface modification of the hydrophobic particles by hydrophilic polymers such as polyethylene glycol would also help to improve biocompatibility and enhance stability of NPs in plasma, although, the modification might also prevent target toxins to be captured by NPs due to steric hindrance.…”

“…However, the combined affinity and specificity of the NPs were not sufficient to detoxify melittin in animal models. Recently, we demonstrated that by applying a molecular imprinting (19) or an affinity purification process (20) together with the combination of functional monomers, NPs with greater affinity to the target peptide was achieved. Furthermore, the NPs with enhanced binding affinity showed neutralization of the toxin in vivo (3).…”

Polymer antidotes for toxin sequestration

“…Isolated NPs containing a greater proportion of high affinity recognition sites may be more effective in future therapeutic applications. Shea et al reported that multifunctional polymer NPs, approximately the size of a large protein complex (30-50 nm), can be "purified" on the basis of toxic peptide affinity just as antibodies, using an affinity chromatography strategy [22]. In the general protocol for affinity purification of proteins, isolated proteins on the solid affinity support are released by selective elution induced by either pH and/or salt gradients or by adding an excess of competing ligand.…”

Preparation of nanogel-immobilized porous gel beads for affinity separation of proteins: fusion of nano and micro gel materials

“…Dansyl methacrylamide was synthesized using the method reported. 22 When the NPs were polymerized with greater than 0.1 mol% of dansyl methacrylamide, a mono-modal size distribution of NPs was not obtained because of the tendency of the NPs to aggregate during the preparation process. When the NPs were polymerized with 0.01 mol% of dansyl methacrylamide, NPs with a mono-modal size distribution were obtained.…”

“…Target affinity can be enhanced by optimizing the feed ratio of functional monomers, 19 by molecular imprinting polymerization 20,21 and/or by affinity purification procedures. 22 It has also been reported that porous membranes polymerized with functional monomers containing carbohydrates can be used as affinity separation media for lectins. [23][24][25] Solid supports for affinity purification media must be carefully designed to achieve an efficient purification process.…”